Cell physiology is characterized by the interplay of numerous metabolic pathways and processes. Integration is essential to create a metabolism that is robust, yet adaptable to complex environmental conditions, such as growth in the presence or absence of oxygen. Although aerobic respiration provides a substantial energetic advantage, it necessarily generates toxic oxygen species that can damage macromolecules (Gonzalez-Flecha, B. and Demple, B., J. Biol. Chem. 270:13681-7, 1995; Imlay, J. A. and Fridovich, I., J. Biol. Chem. 266:6957-65,1991. For example, superoxide radicals (O2−) can oxidize labile [4Fe-4S] to inactive [3Fe-4S] clusters (Flint, D. H., et al., J. Biol. Chem. 268:22369-76, 1993; Kuo, C. F., et al., J. Biol. Chem. 262:4724-7,1987). Such oxidation has at least two detrimental consequences, inactivation of enzymes containing [Fe—S] clusters (Flint, D. H., et al., supra, 1993; Gardner, P. R. and Fridovich, I., Arch. Biochem. Biophys. 284:106-11, 1991; Gardner, P. R. and Fridovich, I., J Biol Chem 266:1478-83,1991; Gardner, P. R. and Fridovich, I., J. Biol. Chem. 266:19328-33,1991), and increased DNA damage (Imlay, J. A. and Linn, S., Science 240:1302-9, 1988; Keyer, K. and Imlay, J. A., Proc. Natl. Acad. Sci. USA 93:13635-40,1996). DNA damage results from ferrous ions, released during the oxidation of [4Fe-4S] clusters. These ions participate in Fenton chemistry (Fe(II)+H2O2+H+→Fe(III)+H2O+OH●), with the hydroxyl radicals damaging DNA and other macromolecules (Keyer, K. and Imlay, J. A., supra, 1996; Liochev, S. I. and Fridovich, I., Free Radic. Biol. Med. 16:29-33, 1994; Srinivasan, C., et al., J. Biol. Chem. 275:29187-92, 2000). It would not be surprising that many cellular anomalies caused by increased superoxide concentration result from oxidization of [Fe—S] clusters (Keyer, K. and Imlay, J. A., supra, 1996).
Several systems exist to reduce the potential for damage by superoxide radicals (Storz, G. and Imlay, J. A., Curr. Opin. Microbiol. 2:188-94, 1999). In general, these systems either prevent the damage from occurring or repair it. The Sox regulon is a good example of the former. This regulon includes a number of genes that are induced under conditions of oxidative stress via the SoxRS regulatory system (Hidalgo, E. and Demple, B., Embo J. 16:1056-65,1997; Gaudu, P., et. al., J. Biol. Chem. 272:5082-6,1997; Liochev, S. I., et al., J. Biol. Chem. 274:9479-81, 1999). One component of this system is the superoxide dismutase enzymes (SOD, EC 1.15.1.1) that catalyze the formation of molecular oxygen and hydrogen peroxide from two superoxide radicals (O2−+O2−+2H+→O2+H2O2). The resulting hydrogen peroxide (H2O2) is a substrate for catalase (hydroperoxidase) enzymes (EC 1.11.1.6,1.11.1.7) that convert it to water and molecular oxygen. A distinct way of protecting [Fe—S] clusters is shown by the FeSII protein of Azotobacter vinelandii. The FeSII, or Shetna protein, forms a complex with nitrogenase under periods of high oxygen exposure, thus protecting the essential [Fe—S] cluster from oxidation (Lou, J., et al., Biochemistry 38:5563-71, 1999; Shethna, Y. I., et al., Biochem. Biophys. Res. Commun. 31:862-8,1968).
In addition to eliminating superoxide per se, mechanisms to repair damage incurred by the superoxide radicals have evolved. This second strategy includes multiple repair systems that are specific for DNA damage (McCullough, A. K., et al., Annu. Rev. Biochem. 68:255-85,1999; Cadet, J., et al., Mutat. Res. 462:121-8, 2000; Boiteux, S. and Radicella, J. P., Biochimie 81:59-67,1999). The DNA glycosylase MutY, which itself contains an [Fe—S] cluster (Michaels, M. L., et al., Nucleic Acids Res. 18:3841-5, 1990; Porello, S. L., et al., Biochemistry 37:6465-75, 1998), recognizes the mispairing of an oxidized guanine base (8-oxo-guanine) with adenine and cleaves the relevant adenine (Michaels, M. L., et al., Biochemistry 31:10964-8, 1992). This cleavage product becomes the target for additional repair enzymes that prevent the generation of a G●C to T●A transversion mutation.
Another example involves direct repair of oxidized [Fe—S] clusters in vivo. The enzyme paradigm for the majority of studies addressing the in vivo and in vitro reconstitution of [Fe—S] clusters is aconitase (Acn, EC 4.2.1.3) (Kennedy, M. C. and Beinert, H., J. Biol. Chem. 263:8194-8, 1988; Gardner, P. R. and Fridovich, I., J. Biol. Chem. 267:8757-63,1992; Gardner, P. R. and Fridovich, I., Arch. Biochem. Biophys. 301:98-102, 1993). Part of the catalytic [4Fe-4S] center in aconitase is exposed to the solution and is not sequestered by the enzyme; thus the enzyme is sensitive to attack by superoxide (Gardner, P. R and Fridovich, I., supra, 1992; Beinert, H., et al., Chem. Rev. 96:2335-2373, 1996). Although extensive work has been preformed to characterize in vitro reactivation of oxidized [Fe—S] clusters (Kennedy, M. C. and Beinert, H., supra, 1988), the participants in [Fe—S] cluster repair in vivo are less well defined (Gardner, P. R. and Fridovich, I., supra, 1993). The benefit of in vivo repair of [Fe—S] clusters is at least two fold, first the restoration of enzyme activity, and second, the decrease of free iron.
Several experiments have suggested that glutathione (GSH) is involved in the in vivo repair and possibly biosynthesis, of the [Fe—S] center in aconitase (Gardner, P. R. and Fridovich, I., supra, 1993). When Escherichia coli strains in vivo were challenged with oxygen total aconitase activity decreased, as expected for an enzyme with a labile [Fe—S] cluster. However, when the oxygen challenge was removed, unlike the wild-type strain, gshA (encodes y-I-glutamyl-I-cysteine synthetase, EC 6.3.2.2) mutants were unable to regain aconitase activity in the absence of protein synthesis (Gardner, P. R. and Fridovich, I., supra, 1993).
Further, gshA mutants of E. coli have reduced total aconitase activity (Gardner, P. R. and Fridovich, I., supra, 1993).
Needed in the art is an improved method of protecting cells and oxygen-labile enzymes from superoxide damage.